Gremlin-1 inhibitor for the treatment of a bone fracture or bone defect

ABSTRACT

The present invention relates to methods for the treatment of a bone fracture or bone defect. The invention discloses the effective use of an anti-gremlin-1 antibody to accelerate the healing and bridging of bone tissue in segmental gap defects; and demonstrates that inhibitors of gremlin-1 activity may provide improved therapies for treating or preventing fracture non-union.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2019/053726, filed Feb. 14, 2019.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jun. 3, 2020 and is 60 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to methods for the treatment of a bonefracture or bone defect. The invention discloses the effective use of ananti-gremlin-1 antibody to accelerate the healing and bridging of bonetissue in segmental gap defects; and demonstrates that inhibitors ofgremlin-1 activity may provide improved therapies for treating orpreventing fracture non-union.

BACKGROUND

A bone fracture is a break or crack in bone tissue and may be the resultof a traumatic injury, such as a fall or impact, but can also occur as aresult of diseases that affect bone integrity.

Non-stabilised bone fractures heal through the process of endochondralossification, which is initiated through the formation of a blood clotor haematoma. This is coupled with an inflammatory response thatmodulates immune cells and surrounding skeletal stem cell populations.The haematoma is subsequently replaced with a mineralised cartilaginouscallus through the action of various growth factors includingtransforming growth factor beta (TGFβ) (Cho et al; 2002), fibroblasticgrowth factors (FGFs) (Schmid et al; 2009), and bone morphogenicproteins (BMPs) (Yu et al; 2010). Through the actions of osteoclasts andosteoblasts, the mineralised callus is replaced by woven bone. The finalremodelling stage involves the replacement of woven bone with lamellarbone. The completion of this process can take many years depending onthe age and disease status of the patient.

Bone fractures are generally treated clinically through stabilisation,via the use of a support such as a splint, cast, or brace. In extremecases involving complex fractures surgical intervention may be requiredand involves the use of internal and external fixators that are attacheddirectly to the bone. Even with these measures, in approximately 10% ofpatients the tissue repair process is deficient (Einhorn et al; 2014)resulting in delayed bone union (failure to reach union 6 monthspost-fracture) or non-union. A non-union is defined as incompletehealing within 9 months, combined with a lack of radiologicalcharacteristics associated with fracture healing being observed overthree consecutive months (Buza et al; 2016). Current surgical techniquesfor repairing non-union fractures and critical bone defects are oftenlimited in terms of quantity and quality of the materials available.Commonly used treatments involve the autologous or allogenic graft,however these carry the additional risk of donor site morbidity (Gouletet al; 1997) and infection (Bostrom et al; 2005), respectively.

A bone defect is a loss of bone, due to trauma or disease.

There is currently a great unmet medical need for improved treatment ofbone fractures and bone defects. Accordingly, it is an object of thepresent invention to provide new methods for the treatment of a bonefracture or bone defect.

The present invention provides inhibitors of gremlin-1 activity for usein the treatment of a bone fracture or bone defect. The inventiondiscloses the effective use of an anti-gremlin-1 antibody to acceleratethe healing and bridging of bone tissue in segmental gap defects; anddemonstrates that inhibitors of gremlin-1 activity may provide improvedtherapies for treating or preventing fracture non-union.

DESCRIPTION OF THE INVENTION

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of skill in the art.All publications referred to herein are incorporated by reference.

It will be appreciated that any of the embodiments described herein maybe combined.

The present invention provides an inhibitor of gremlin-1 activity foruse in the treatment of a bone fracture or bone defect. The inventionalso provides the use of an inhibitor of gremlin-1 activity for themanufacture of a medicament for the treatment of a bone fracture or bonedefect. The invention further provides a method for the treatment of abone fracture or bone defect comprising administering a therapeuticallyeffective amount of an inhibitor of gremlin-1 activity.

Gremlin-1 (also known as Drm and CKTSF1B1) is a 184 amino acidglycoprotein which forms part of the DAN family of cysteine-knotsecreted proteins (along with Cerberus and Dan amongst others). Gremlinbinds and inhibits the ability of BMP-2, 4, and 7 to signal along with adocumented pro-angiogenic role possibly through agonism of VEGFR2. Themain role of Gremlin-1 is during development, in which it is vitalduring kidney formation and during limb bud formation.

Bone morphogenetic protein (BMP) signalling is known to controlendochondral bone formation, with Gremlin 1 (GREM1) being one of thenatural antagonists of this pathway through its binding to BMP2, BMP4and BMP7 (Hsu et al; 1998). GREM1 conditional deletion in osteoblastsresults in sensitisation of BMP signalling/activity and enhanced boneformation in vivo (Gazzerro et al; 2007), whilst conditionaloverexpression in the same cell type causes osteopenia and spontaneousfractures (Gazzerro et al; 2005). Furthermore, although the globalknockout is embryonic lethal in a BL6 background, 49% of pups survivedlonger than 24 hrs post birth in the C57BL/6/FVB mixed geneticbackground, and whilst developmental skeletal defects were abundantlypresent, elevated bone formation rates could be observed (Canalis et al;2012). Despite this developmental function of GREM1 there is no data tosuggest that inhibition of this protein alone will enhance postnatalbone fracture repair. Indeed, although endochondral bone formation isthe main mechanism of skeletogeneis at embryonic stages, the mechanismsthat regulate cell recruitment are distinct processes when compared topostnatal fracture repair (Ferguson et al; 1999). The role ofinflammation has been indicated as a key factor in adult bone repair,thus developmental factors controlling skeletogenesis processes cannotsimply be extrapolated to postnatal repair mechanisms.

The term Gremlin-1 as used in the present invention typically has thesequence as set out in the UniProt entry 060565 (SEQ ID NO: 1). The termGremlin-1 may also refer to a Gremlin-1 polypeptide which:

(a) comprises or consists of the amino acid sequence of SEQ ID NO: 1with or without the N-terminal signal peptide, i.e. may comprise orconsist of the mature peptide sequence as shown in SEQ ID NO: 21; or

(b) is a derivative having one or more amino acid substitutions,modifications, deletions or insertions relative to the amino acidsequence of SEQ ID NO: 1 with or without the N-terminal signal peptide(as shown in SEQ ID NO: 21), which retains the activity of Gremlin-1,such as the amino acid sequence of SEQ ID NO: 20.

(c) a variant thereof, such variants typically retain at least about60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or 95% identity to SEQ ID NO: 1(or SEQ ID NO: 20 or 21) (or even about 96%, 97%, 98% or 99% identity).In other words, such variants may retain about 60% -about 99% identityto SEQ ID NO: 1, suitably about 80%-about 99% identity to SEQ ID NO: 1,more suitably about 90%-about 99% identity to SEQ ID NO: 1 and mostsuitably about 95%-about 99% identity to SEQ ID NO: 1. Variants aredescribed further below.

As discussed further below, residue numbers are typically quoted basedon the sequence of SEQ ID NO: 1. However, residue numbering couldreadily be extrapolated by the skilled person to a derivative or variantsequence as discussed above. Where residue numbers are quoted, theinvention also encompasses these residues on a variant or derivativesequence.

The present inventors have crystallised human Gremlin-1 alone, and incomplex with an antibody termed Ab 7326 (Fab fragments). Crystallisationof Gremlin-1 has allowed putative residues in the BMP binding site to bedetermined. Furthermore, crystallisation with Ab 7326, which is anallosteric inhibitory antibody, has allowed residues in the antibodyepitope to be determined. (WO 2018/115017 A2). Antibodies binding thisepitope have potential as therapeutic agents in the treatment of a bonefracture or bone defect.

Inhibitors of Gremlin-1 activity

An inhibitor of gremlin-1 activity according to the present invention isan agent that reduces or blocks the activity of gremlin-1. Inhibitorsaccording to the present invention may partially or completely inhibitgremlin-1 activity. Inhibitors of use in the present invention includewithout limitation, inhibitors that are capable of binding to gremlin-1or to a nucleic acid molecule encoding gremlin-1, or are capable ofinhibiting the expression of gremlin-1. Such inhibitors may be, withoutlimitation, proteins, polypeptides, peptides, peptidomimetics, nucleicacids (e.g. DNA, RNA, antisense RNA and siRNA), carbohydrates, lipids,and small molecules.

In one embodiment, the inhibitor of gremlin-1 activity is ananti-gremlin-1 antibody or a functionally active fragment, variant orderivative thereof.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An antibody or immunoglobulin typically refers to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen-binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or V_(H)) and a heavy chain constant region.Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR).

The constant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

An antibody for use in the present invention may be a monoclonalantibody or a polyclonal antibody, and will typically be a monoclonalantibody. An antibody for use in the invention may be a chimericantibody, a CDR-grafted antibody, a nanobody, a human or humanisedantibody or an antigen-binding portion of any thereof.

Polyclonal antibodies may be produced by routine methods, such asimmunisation of a suitable animal with the antigen of interest. Bloodmay be subsequently removed from the animal and the immunoglobulinfraction purified.

Antibodies against Gremlin-1 may be obtained, where immunisation of ananimal is necessary, by administering the polypeptides to an animal,e.g. a non-human animal, using well-known and routine protocols, see forexample Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4,Blackwell Scientific Publishers, Oxford, England, 1986). Manywarm-blooded animals, such as rabbits, mice, rats, sheep, goats, cows,camels, llamas or pigs may be immunised. However, rabbits, mice, andrats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy,pp77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by for example the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO 92/02551; WO 2004/051268 and WO 2004/106377.

The antibodies for use in the present invention can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

Fully human antibodies are those antibodies in which the variableregions and the constant regions (where present) of both the heavy andthe light chains are all of human origin, or substantially identical tosequences of human origin, but not necessarily from the same antibody.Examples of fully human antibodies may include antibodies produced, forexample by the phage display methods described above and antibodiesproduced by mice in which the murine immunoglobulin variable andoptionally the constant region genes have been replaced by their humancounterparts e.g. as described in general terms in EP 0546073, U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,EP 0438474 and EP 0463151.

Alternatively, an antibody according to the invention may be produced bya method comprising immunising a non-human mammal with a Gremlin-1immunogen; obtaining an antibody preparation from said mammal; derivingtherefrom monoclonal antibodies that recognise Gremlin-1.

The antibody molecules for use in the present invention may comprise acomplete antibody molecule having full length heavy and light chains ora fragment or antigen-binding portion thereof. The term “antigen-bindingportion” of an antibody refers to one or more fragments of an antibodythat retain the ability to selectively bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. The antibodies and fragments andantigen binding portions thereof may be, but are not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)₂, Fv, single domain antibodies(e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies,Bis-scFv, diabodies, triabodies, tetrabodies and epitope-bindingfragments of any of the above (see for example Holliger and Hudson,2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, DrugDesign Reviews-Online 2(3), 209-217). The methods for creating andmanufacturing these antibody fragments are well known in the art (seefor example Verma et al., 1998, Journal of Immunological Methods, 216,165-181). Other antibody fragments for use in the present inventioninclude the Fab and Fab′ fragments described in International patentapplications WO 2005/003169, WO 2005/003170 and WO 2005/003171 andFab-dAb fragments described in International patent application WO2009/040562. Multi-valent antibodies may comprise multiple specificitiesor may be monospecific (see for example WO 92/22853 and WO 2005/113605).These antibody fragments may be obtained using conventional techniquesknown to those of skill in the art, and the fragments may be screenedfor utility in the same manner as intact antibodies.

In one example, the functionally active antibody fragment for use in thepresent invention is a Fab, Fab′, F(ab′)2, Fv or scFv.

The constant region domains of the antibody molecule for use in thepresent invention, if present, may be selected having regard to theeffector functions which may be required. For example, the constantregion domains may be human IgA, IgD, IgE, IgG or IgM domains. Inparticular, human IgG constant region domains may be used, especially ofthe IgG1 and IgG3 isotypes when antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotypes may be used whenantibody effector functions are not required. In one example, theisotype is IgG4P, as described by Angal S. et al, Mol Immunol, Vol30(1), p105-108, 1993.

An antibody for use in the invention may be prepared, expressed, createdor isolated by recombinant means, such as (a) antibodies isolated froman animal (e.g., a mouse) that is transgenic or transchromosomal for theimmunoglobulin genes of interest or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibodyof interest, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of immunoglobulin gene sequences to other DNA sequences.

An antibody for use in the invention may be a human antibody or ahumanised antibody. The term “human antibody”, as used herein, isintended to include antibodies having variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. Human antibodies for use in the invention may include aminoacid residues not encoded by human germline immunoglobulin sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo). However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma that includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “derivative” refers to any modified form of the antibody, forexample a conjugate of the antibody and another agent or effectormolecule.

An effector molecule may comprise a single effector molecule or two ormore such molecules so linked as to form a single moiety that can beattached to the antibodies for use in the present invention. Where it isdesired to obtain an antibody fragment linked to an effector molecule,this may be prepared by standard chemical or recombinant DNA proceduresin which the antibody fragment is linked either directly or via acoupling agent to the effector molecule. Techniques for conjugating sucheffector molecules to antibodies are well known in the art (see,Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al.,eds., 1987, pp. 623-53; Thorpe et al., 1982 , Immunol. Rev., 62:119-58and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).Particular chemical procedures include, for example, those described inWO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 2003/031581.Alternatively, where the effector molecule is a protein or polypeptidethe linkage may be achieved using recombinant DNA procedures, forexample as described in WO 86/01533 and EP 0392745.

The effector molecule may increase the half-life of the antibody invivo, and/or reduce immunogenicity of the antibody and/or enhance thedelivery of an antibody across an epithelial barrier to the immunesystem. Examples of suitable effector molecules of this type includepolymers, albumin, albumin binding proteins or albumin binding compoundssuch as those described in WO 2005/117984.

The term “humanised antibody” is intended to refer to CDR-graftedantibody molecules in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Additional framework region modifications may bemade within the human framework sequences.

As used herein, the term ‘CDR-grafted antibody molecule’ refers to anantibody molecule wherein the heavy and/or light chain contains one ormore CDRs (including, if desired, one or more modified CDRs) from adonor antibody (e.g. a murine or rat monoclonal antibody) grafted into aheavy and/or light chain variable region framework of an acceptorantibody (e.g. a human antibody). For a review, see Vaughan et al,Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather thanthe entire CDR being transferred, only one or more of the specificitydetermining residues from any one of the CDRs described herein above aretransferred to the human antibody framework (see for example, Kashmiriet al., 2005, Methods, 36, 25-34). In one embodiment only thespecificity determining residues from one or more of the CDRs describedherein above are transferred to the human antibody framework. In anotherembodiment only the specificity determining residues from each of theCDRs described herein above are transferred to the human antibodyframework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Suitably, the CDR-grafted antibody for use in the present invention hasa variable domain comprising human acceptor framework regions as well asone or more of the CDRs or specificity determining residues describedabove. Thus, provided in one embodiment is a neutralising CDR-graftedantibody wherein the variable domain comprises human acceptor frameworkregions and non-human donor CDRs.

Examples of human frameworks which can be used in the present inventionare KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available for example at: Worldwide Website:vbase2.org/ (see Retter et al, Nucl. Acids Res. (2005) 33 (supplement1), D671-D674).

In a CDR-grafted antibody for use in the present invention, the acceptorheavy and light chains do not necessarily need to be derived from thesame antibody and may, if desired, comprise composite chains havingframework regions derived from different chains.

Also, in a CDR-grafted antibody for use in the present invention, theframework regions need not have exactly the same sequence as those ofthe acceptor antibody. For instance, unusual residues may be changed tomore frequently occurring residues for that acceptor chain class ortype. Alternatively, selected residues in the acceptor framework regionsmay be changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al., 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of posttranslational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995).

In one embodiment the antibody heavy chain comprises a CH1 domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

Biological molecules, such as antibodies or fragments, contain acidicand/or basic functional groups, thereby giving the molecule a netpositive or negative charge. The amount of overall “observed” chargewill depend on the absolute amino acid sequence of the entity, the localenvironment of the charged groups in the 3D structure and theenvironmental conditions of the molecule. The isoelectric point (pI) isthe pH at which a particular molecule or surface carries no netelectrical charge. In one embodiment the antibody or fragment accordingto the present disclosure has an isoelectric point (pI) of at least 7.In one embodiment the antibody or fragment has an isoelectric point ofat least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pI ofthe antibody is 8. Programs such as ** ExPASY (Worldwide Website:expasy.ch/tools/pi_tool.html) (see Walker, The Proteomics ProtocolsHandbook, Humana Press (2005), 571-607) may be used to predict theisoelectric point of the antibody or fragment.

Antibodies for use in the invention may comprise at least one, at leasttwo or all three heavy chain CDR sequences of SEQ ID NOS: 4 to 6(HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3sequences of the Ab7326 antibody of the Examples as determined usingKabat methodology.

The Kabat and Chothia methods for determining CDR sequences are wellknown in the art (as well as other techniques). CDR sequences may bedetermined using any appropriate method and in the present invention,whilst Kabat is typically employed, other techniques could be used aswell. In the present instance, SEQ ID NO: 3 presents the Ab7326 HCDR1sequence as determined using a combined Chothia & Kabat definition.

Antibodies for use in the invention may comprise at least one, at leasttwo or all three light chain CDR sequences of SEQ ID NOS: 7 to 9(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3sequences of Ab7326 using Kabat methodology.

In one embodiment, the antibody comprises at least a HCDR3 sequence ofSEQ ID NO: 6.

Typically, the antibody comprises at least one heavy chain CDR sequenceselected from SEQ ID NOS: 4 to 6 and at least one light chain CDRsequence selected from SEQ ID NOS 7 to 9. The antibody may comprise atleast two heavy chain CDR sequences selected from SEQ ID NOS: 4 to 6 andat least two light chain CDR sequences selected from SEQ ID NOS: 7 to 9.The antibody typically comprises all three heavy chain CDR sequences ofSEQ ID NOS: 4 to 6 (HCDR1/HCDR2/HCDR3 respectively) and all three lightchain CDR sequences SEQ ID NOS: 7 to 9 (LCDR1/LCDR2/LCDR3 respectively).The antibodies may be chimeric, human or humanised antibodies.

The antibody may comprise a heavy chain variable region (HCVR) sequenceof SEQ ID NO: 10 or 12 (the HCVR of Ab7326 variants 1 and 2). Theantibody may comprise a light chain variable region (LCVR) sequence ofSEQ ID NO: 11 or 13 (the LCVR of Ab7326 variants 1 and 2). The antibodypreferably comprises the heavy chain variable region sequence of SEQ IDNO: 10 or 12 and the light chain variable region sequence of SEQ ID NO:11 or 13 (especially HCVR/LVCR pairs of SEQ ID NOs: 10/11 or 12/13).

Ab7326 variants 1 and 2 differ by a single amino acid in the heavy chainvariable region, and by a single amino acid in the light chain variableregion, as follows:

-   -   Heavy chain variable region variant 1 has glutamic acid (E) at        position 6. (SEQ ID NO:10)    -   Heavy chain variable region variant 2 has glutamine (Q) at        position 6. (SEQ ID NO:12)    -   Light chain variable region variant 1 has serine (S) at        position 7. (SEQ ID NO:11)    -   Light chain variable region variant 2 has threonine (T) at        position 7. (SEQ ID NO:13)

Thus, in one embodiment, the antibody comprises a heavy chain variableregion (HCVR) sequence of SEQ ID NO: 10, wherein the glutamic acidresidue at position 6 is substituted with a glutamine residue (E6Q);wherein the residue numbering is according to SEQ ID NO: 10.

In one embodiment, the antibody comprises a heavy chain variable region(HCVR) sequence of SEQ ID NO: 12, wherein the glutamine residue atposition 6 is substituted with a glutamic acid residue (Q6E); whereinthe residue numbering is according to SEQ ID NO: 12.

In one embodiment, the antibody comprises a light chain variable region(LCVR) sequence of SEQ ID NO: 11, wherein the serine residue at position7 is substituted with a threonine residue (S7T); wherein the residuenumbering is according to SEQ ID NO: 11.

In one embodiment, the antibody comprises a light chain variable region(LCVR) sequence of SEQ ID NO: 13, wherein the threonine residue atposition 7 is substituted with a serine residue (T7S); wherein theresidue numbering is according to SEQ ID NO: 13.

In one embodiment, the antibody comprises the sequence of SEQ ID NO: 3or 4 for HCDR1, the sequence of SEQ ID NO: 5 for HCDR2, the sequence ofSEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1, thesequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 forLCDR3; and wherein the heavy chain variable region comprises a sequencehaving at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99%identity), to the sequence of SEQ ID NO: 10 and the light chain variableregion comprises a sequence having at least 95% identity, (e.g. at least95%, 96%, 97%, 98% or 99% identity), to the sequence of SEQ ID NO: 11.

In one embodiment, the antibody comprises the sequence of SEQ ID NO: 3or 4 for HCDR1, the sequence of SEQ ID NO: 5 for HCDR2, the sequence ofSEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1, thesequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 forLCDR3; and wherein the heavy chain variable region comprises a sequencehaving at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99%identity), to the sequence of SEQ ID NO: 12 and the light chain variableregion comprises a sequence having at least 95% identity, (e.g. at least95%, 96%, 97%, 98% or 99% identity) to the sequence of SEQ ID NO: 13.

The antibody may comprise a heavy chain (H-chain) sequence of

SEQ ID NO: 14 mouse full length IgG1 heavy chain variant 1, or

SEQ ID NO: 28 mouse full length IgG1 heavy chain variant 2, or

SEQ ID NO: 30 human full length IgG1 heavy chain variant 1, or

SEQ ID NO: 16 human full length IgG1 heavy chain variant 2, or

SEQ ID NO: 22 human full length IgG4P heavy chain variant 1, or

SEQ ID NO: 34 human full-length IgG4P heavy chain variant 2, or

SEQ ID NO: 18 Fab heavy chain variant 1, or

SEQ ID NO: 32 Fab heavy chain variant 2.

The antibody may comprise a light chain (L-chain) sequence of

SEQ ID NO: 15 mouse full length IgG1 light chain variant 1, or

SEQ ID NO: 29 mouse full length IgG1 light chain variant 2, or

SEQ ID NO: 31 human full length IgG1 light chain variant 1, or

SEQ ID NO: 17 human full length IgG1 light chain variant 2, or

SEQ ID NO: 23 human full length IgG4P light chain variant 1, or

SEQ ID NO: 35 human full-length IgG4P light chain variant 2, or

SEQ ID NO: 19 Fab light chain variant 1, or

SEQ ID NO: 33 Fab light chain variant 2.

In one example, the antibody comprises a heavy chain/light chainsequence pair of

SEQ ID NOs: 14/15 mouse full length IgG1 variant 1, or

SEQ ID NOs: 28/29 mouse full length IgG1 variant 2, or

SEQ ID NOs: 30/31 human full length IgG1 variant 1, or

SEQ ID NOs: 16/17 human full length IgG1 variant 2, or

SEQ ID NOs: 22/23 human full length IgG4P variant 1, or

SEQ ID NOs: 34/35 human full-length IgG4P variant 2, or

SEQ ID NOs: 18/19 Fab light chain variant 1, or

SEQ ID NOs: 32/33 Fab light chain variant 2.

The variant forms of corresponding sequences may be interchanged. Forexample, the antibody may comprise a heavy chain/light chain sequencepair of

SEQ ID NOs: 14/29 mouse full length IgG1 heavy chain variant 1/lightchain variant 2, or

SEQ ID NOs: 28/15 mouse full length IgG1 heavy chain variant 2/lightchain variant 1, or

SEQ ID NOs: 30/17 human full length IgG1 heavy chain variant 1/lightchain variant 2, or

SEQ ID NOs: 16/31 human full length IgG1 heavy chain variant 2/lightchain variant 1, or

SEQ ID NOs: 22/35 human full length IgG4P heavy chain variant 1/lightchain variant 2, or

SEQ ID NOs: 34/23 human full-length IgG4P heavy chain variant 2/lightchain variant 1, or

SEQ ID NOs: 18/33 Fab heavy chain variant 1/light chain variant 2, or

SEQ ID NOs: 32/19 Fab heavy chain variant 2/light chain variant 1.

The antibodies may be chimeric, human or humanised antibodies.

The antibody may alternatively be or may comprise a variant of one ofthe specific sequences recited above. For example, a variant may be asubstitution, deletion or addition variant of any of the above aminoacid sequences.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20 ormore (typically up to a maximum of 50) amino acid substitutions and/ordeletions from the specific sequences discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Substitution” variants typicallyinvolve the replacement of one or more amino acids with the same numberof amino acids and making conservative amino acid substitutions. Forexample, an amino acid may be substituted with an alternative amino acidhaving similar properties, for example, another basic amino acid,another acidic amino acid, another neutral amino acid, another chargedamino acid, another hydrophilic amino acid, another hydrophobic aminoacid, another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

TABLE 1 Amino acid properties. Ala aliphatic, hydrophobic, neutral Methydrophobic, neutral Cys polar, hydrophobic, neutral Asn polar,hydrophilic, neutral Asp polar, hydrophilic, charged (−) Prohydrophobic, neutral Glu polar, hydrophilic, charged (−) Gln polar,hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar,hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, hydrophilic, Thr polar, hydrophilic,neutral charged (+) Ile aliphatic, hydrophobic, neutral Val aliphatic,hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic,hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

“Derivatives” or “variants” generally include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post- production modification, or whenthe antibody is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/orligation of nucleic acids.

Variant antibodies may have an amino acid sequence which has more thanabout 60%, or more than about 70%, e.g. 75 or 80%, typically more thanabout 85%, e.g. more than about 90 or 95% amino acid identity to theamino acid sequences disclosed herein (particularly the HCVR/LCVRsequences and the H- and L-chain sequences). Furthermore, the antibodymay be a variant which has more than about 60%, or more than about 70%,e.g. 75 or 80%, typically more than about 85%, e.g. more than about 90or 95% amino acid identity to the HCVR/LCVR sequences and the H- andL-chain sequences disclosed herein, whilst retaining the exact CDRsdisclosed for these sequences. Variants may retain at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the HCVR/LCVRsequences and to the H- and L-chain sequences disclosed herein (in somecircumstances whilst retaining the exact CDRs).

Variants typically retain about 60% -about 99% identity, about 80%-about99% identity, about 90%-about 99% identity or about 95%-about 99%identity. This level of amino acid identity may be seen across the fulllength of the relevant SEQ ID NO sequence or over a part of thesequence, such as across about 20, 30, 50, 75, 100, 150, 200 or moreamino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatized.

Antibodies having specific sequences and derivatives and variants whichmaintain the function or activity of these chains are therefore providedfor use in the present invention.

“Derivatives” as used herein is intended to include reactivederivatives, for example thiol-selective reactive groups such asmaleimides. The reactive group may be linked directly or through alinker segment to a polymer. It will be appreciated that the residue ofsuch a group will in some instances form part of the product as thelinking group between the antibody fragment and the polymer.

The polymer may be a synthetic or a naturally occurring polymer, forexample an optionally substituted straight or branched chainpolyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched orunbranched polysaccharide, e.g. a homo- or hetero-polysaccharide.

Specific optional substituents which may be present on a syntheticpolymer include one or more hydroxy, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran,glycogen or derivatives thereof.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50000 Da, forexample from 5000 to 40000 Da such as from 20000 to 40000 Da. Thepolymer size may in particular be selected on the basis of the intendeduse of the product for example ability to localize to certain tissues orextend circulating half-life (for review see Chapman, 2002, AdvancedDrug Delivery Reviews, 54, 531-545). Thus, for example, where theproduct is intended to leave the circulation and penetrate tissue, itmay be advantageous to use a small molecular weight polymer, for examplewith a molecular weight of around 5000 Da. For applications where theproduct remains in the circulation, it may be advantageous to use ahigher molecular weight polymer, for example having a molecular weightin the range from 20000 Da to 40000 Da.

Suitable polymers include a polyalkylene polymer, such as apoly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or aderivative thereof, and especially with a molecular weight in the rangefrom about 15000 Da to about 40000 Da.

In one example antibodies for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods (see for example U.S. Pat. Nos.5,219,996; 5,667,425; WO98/25971, WO2008/038024). In one example theantibody molecule is a modified Fab fragment wherein the modification isthe addition to the C-terminal end of its heavy chain one or more aminoacids to allow the attachment of an effector molecule. Suitably, theadditional amino acids form a modified hinge region containing one ormore cysteine residues to which the effector molecule may be attached.Multiple sites can be used to attach two or more PEG molecules.

Antibodies may compete for binding to Gremlin-1 with, or bind to thesame epitope as, those defined above in terms of H-chain/L-chain,HCVR/LCVR or CDR sequences. In particular, an antibody may compete forbinding to Gremlin-1 with, or bind to the same epitope as, an antibodywhich comprises a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequencecombination of SEQ ID NOs: 4/5/6/7/8/9. An antibody may compete forbinding to Gremlin-1 with, or bind to the same epitope as, an antibodywhich comprises a HCVR and LCVR sequence pair of SEQ ID NOs: 10/11 or12/13 or full length chains of SEQ ID Nos: 14/15 or 16/17.

An “epitope” is a region of an antigen that is bound by an antibody.Epitopes may be defined as structural or functional. Functional epitopesare generally a subset of the structural epitopes and have thoseresidues that directly contribute to the affinity of the interaction.Epitopes may also be conformational, that is, composed of non-linearamino acids. In certain embodiments, epitopes may include determinantsthat are chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference antibody by using routinemethods known in the art. For example, to determine if a test antibodybinds to the same epitope as a reference antibody for use in theinvention, the reference antibody is allowed to bind to a protein orpeptide under saturating conditions. Next, the ability of a testantibody to bind to the protein or peptide is assessed. If the testantibody is able to bind to the protein or peptide following saturationbinding with the reference antibody, it can be concluded that the testantibody binds to a different epitope than the reference antibody. Onthe other hand, if the test antibody is not able to bind to protein orpeptide following saturation binding with the reference antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference antibody of the invention.

To determine if an antibody competes for binding with a referenceantibody, the above-described binding methodology is performed in twoorientations. In a first orientation, the reference antibody is allowedto bind to a protein/peptide under saturating conditions followed byassessment of binding of the test antibody to the protein/peptidemolecule. In a second orientation, the test antibody is allowed to bindto the protein/peptide under saturating conditions followed byassessment of binding of the reference antibody to the protein/peptide.If, in both orientations, only the first (saturating) antibody iscapable of binding to the protein/peptide, then it is concluded that thetest antibody and the reference antibody compete for binding to theprotein/peptide. As will be appreciated by the skilled person, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50%, 75%, 90% or even 99% as measuredin a competitive binding assay (see, e.g., Junghans et al., Cancer Res,1990:50:1495-1502). Alternatively, two antibodies have the same epitopeif essentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies have overlapping epitopes if some amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

The anti-gremlin-1 antibody of the Examples, Ab7326, has been found tobind the following residues of Gremlin-1: Ile131, Lys147, Lys148,Phe149, Thr150, Thr151, Arg169, Lys174 and GIn175; where Lys147, Lys148,Phe149, Thr150, Thr151, Arg169, Lys174 and GIn175 are present on oneGremlin-1 monomer and Ile131 is present on the second Gremlin-1 monomer.The numbering is based on the UniProt entry O60565 of SEQ ID NO: 1. Asdiscussed in the Examples section, these epitope residues wereidentified using NCONT analysis at 4 A from the Gremlin-1-Ab7326 Fabcomplex.

Antibodies for use in the invention may therefore bind to an epitopewhich comprises at least one residue selected from Ile131, Lys147,Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and GIn175 (with residuenumbering based on SEQ ID NO: 1). Antibodies for use in the inventionmay bind an epitope which comprises 2, 3, 4, 5, 6, 7, 8 or all 9 ofthese residues (preferably at least 5 residues).

Antibodies for use in the invention may also recognise an epitope whereIle131 is present on a different Gremlin-1 monomer to the otherresidues.

Although these residues are provided for a particular sequence of humanGremlin-1, the skilled person could extrapolate the positions of theseresidues to other corresponding Gremlin sequences using routinetechniques. Antibodies binding to epitopes comprising the correspondingresidues within these other Gremlin sequences are therefore alsoprovided for use in the invention.

To screen for antibodies that bind to a particular epitope, a routinecross-blocking assay such as that described in Antibodies, Harlow andLane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.) can beperformed. Other methods include alanine scanning mutants, peptide blots(Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavageanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Protein Science 9: 487-496). Such methods are well known in theart.

Antibody epitopes may also be determined by x-ray crystallographyanalysis. Antibodies for use in the present invention may therefore beassessed through x-ray crystallogray analysis of the antibody bound toGremlin-1. Epitopes may, in particular, be identified in this way bydetermining residues on Gremlin-1 within 4A of an antibody paratoperesidue.

Antibodies can be tested for binding to Gremlin-1 by, for example,standard ELISA or Western blotting. An ELISA assay can also be used toscreen for hybridomas that show positive reactivity with the targetprotein. The binding selectivity of an antibody may also be determinedby monitoring binding of the antibody to cells expressing the targetprotein, for example by flow cytometry. Thus, a screening method maycomprise the step of identifying an antibody that is capable of bindingGremlin-1 by carrying out an ELISA or Western blot or by flow cytometry.

Antibodies may selectively (or specifically) recognise Gremlin-1. Anantibody, or other compound, “selectively binds” or “selectivelyrecognises” a protein when it binds with preferential or high affinityto the protein for which it is selective but does not substantiallybind, or binds with low affinity, to other proteins. The selectivity ofan antibody may be further studied by determining whether or not theantibody binds to other related proteins as discussed above or whetherit discriminates between them. Antibodies for use in the inventiontypically recognise human Gremlin-1.

Antibodies may also have cross-reactivity for related proteins, or forhuman Gremlin-1 and for Gremlin-1 from other species.

By specific (or selective), it will be understood that the antibodybinds to the protein of interest with no significant cross-reactivity toany other molecule. Cross-reactivity may be assessed by any suitablemethod described herein. Cross-reactivity of an antibody may beconsidered significant if the antibody binds to the other molecule atleast about 5%, 10′)/0, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to theprotein of interest. An antibody that is specific (or selective) maybind to another molecule at less than about 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that itbinds to the protein of interest. The antibody may bind to the othermolecule at less than about 20%, less than about 15%, less than about10% or less than about 5%, less than about 2% or less than about 1% thestrength that it binds to the protein of interest.

Thus, antibodies suitable for use in the present invention may have ahigh affinity binding for (human) Gremlin-1. The antibody may have adissociation constant (K_(D)) of less than <1 nM, and preferably <500pM. In one example, the antibody has a dissociation constant (K_(D)) ofless than 200 pM. In one example, the antibody has a dissociationconstant (K_(D)) of less than 100 pM. A variety of methods can be usedto determine the binding affinity of an antibody for its target antigensuch as surface plasmon resonance assays, saturation assays, orimmunoassays such as ELISA or RIA, as are well known to persons of skillin the art. An exemplary method for determining binding affinity is bysurface plasmon resonance analysis on a BIAcore™ 2000 instrument(Biacore AB, Freiburg, Germany) using CM5 sensor chips, as described byKrinner et al., (2007) Mol. Immunol. February; 44 (5):916-25. (Epub 2006May 11)).

The anti-Gremlin-1 antibody of the Examples, Ab7326, is an allostericinhibitor of Gremlin-1 activity which binds to an epitope distal fromthe BMP binding site. (WO 2018/115017 A2) Ab7326 binds to Gremlin-1 withexceptionally high affinity with a Kd value<100pM, and is expected to beparticularly useful for use in the present invention.

An inhibitor of gremlin-1 activity may have an effect on any of thefunctions of Gremlin-1, but typically reduces binding of Gremlin-1 toBMP (BMP 2, 4, and/or 7). Gremlin-1 is a negative regulator of BMP andso reduced binding increases signalling through BMP. BMP binding andsignalling may be detected by any method known in the art. The Examplesof the present application describe two functional assays for testingwhether an agent reduces binding of gremlin-1 to BMP. Example 3describes an Idi reporter gene assay, where the Id1 gene is a targetgene of BMP signalling. An increase in the signal in this assay may beused to determine if an agent reduces Gremlin-1 binding to BMP. Example5 describes a SMAD phosphorylation assay. SMAD1, 5 and 8 arephosphorylated upon BMP signalling. An increase in SMAD phosphorylationmay therefore be used to determine whether an agent reduces binding ofGremlin-1 to BMP.

Once a suitable antibody has been identified and selected, the aminoacid sequence of the antibody may be identified by methods known in theart. The genes encoding the antibody can be cloned using degenerateprimers. The antibody may be recombinantly produced by routine methods.

Examples of DNA sequences encoding full length heavy chains and lightchains of Ab7326 are provided in the sequence listing:

-   -   SEQ ID NO: 24 (Human IgG1 heavy chain DNA variant 1)    -   SEQ ID NO: 25 (Human IgG1 light chain DNA variant 1)    -   SEQ ID NO: 26 (Human IgG4P heavy chain DNA variant 1)    -   SEQ ID NO: 27 (Human IgG4P light chain DNA variant 1).

Pharmaceutical Compositions, Dosages and Dosage Regimes

An inhibitor of gremlin-1 activity for use in the present invention maybe provided in a pharmaceutical composition. The pharmaceuticalcomposition will normally be sterile and will typically include apharmaceutically acceptable carrier and/or adjuvant. A pharmaceuticalcomposition for use in the invention may additionally comprise apharmaceutically acceptable adjuvant and/or carrier.

The pharmaceutical compositions for use in the invention may include oneor more pharmaceutically acceptable salts. A “pharmaceuticallyacceptable salt” refers to a salt that retains the desired biologicalactivity of the parent molecule and does not impart any undesiredtoxicological effects. Examples of such salts include acid additionsalts and base addition salts.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier may be suitable for parenteral,e.g. intravenous, intramuscular, intradermal, intraocular,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Alternatively, thecarrier may be suitable for non-parenteral administration, such as atopical, epidermal or mucosal route of administration. The carrier maybe suitable for oral administration. Depending on the route ofadministration, the inhibitor may be coated in a material to protect itfrom the action of acids and other natural conditions that mayinactivate the inhibitor.

Pharmaceutically acceptable carriers comprise aqueous carriers ordiluents. Examples of suitable aqueous carriers that may be employed inthe pharmaceutical compositions for use in the invention include water,buffered water and saline. Examples of other carriers include ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. In many cases,it will be desirable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, micro-emulsion, liposome, or other orderedstructure suited to high drug concentration.

Pharmaceutical compositions for use in the invention may compriseadditional active ingredients.

Also envisaged are kits comprising an inhibitor of gremlin-1 activityand instructions for use in a method of treatment according to theinvention.

Agents for use in the invention or formulations or compositions thereofmay be administered for therapeutic and/or prophylactic treatments.

In therapeutic applications, agents are administered to a subjectalready suffering from a disorder or condition, in an amount sufficientto cure, alleviate or partially arrest the condition or one or more ofits symptoms. Such therapeutic treatment may result in a decrease inseverity of symptoms, or an increase in frequency or duration ofsymptom-free periods. An amount adequate to accomplish this is definedas a “therapeutically effective amount”.

In prophylactic applications, agents are administered to a subject atrisk of a disorder or condition, in an amount sufficient to prevent orreduce the subsequent effects of the condition or one or more of itssymptoms. An amount adequate to accomplish this is defined as a“prophylactically effective amount”. Effective amounts for each purposewill depend on the severity of the disease or injury as well as theweight and general state of the subject.

A subject for administration may be a human or non-human animal. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, dogs, cats, horses, sheep,cows, chickens, amphibians, reptiles, etc. Administration to humans istypical.

An agent or pharmaceutical composition for use in the invention may beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Examples of routes of administrationfor agents or pharmaceutical compositions for use in the inventioninclude parenteral routes, such as intravenous, intramuscular,intradermal, intraocular, intraperitoneal, subcutaneous, or spinalroutes of administration, for example by injection or infusion.Alternatively, an agent or pharmaceutical composition can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration. The agent or pharmaceutical compositionmay be for oral administration.

A suitable dosage of an inhibitory agent or pharmaceutical compositionfor use in the invention may be determined by a skilled medicalpractitioner. Actual dosage levels of the active ingredients in thepharmaceutical compositions for use in the present invention may bevaried so as to obtain an amount of the active ingredient that iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A suitable dose may be, for example, in the range of from about 0.01μg/kg to about 1000 mg/kg body weight, typically from about 0.1 μg/kg toabout 100 mg/kg body weight, of the patient to be treated. For example,a suitable dosage may be from about 1 μg/kg to about 10 mg/kg bodyweight per day or from about 10 μg/kg to about 5 mg/kg body weight perday.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single dose may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by thetherapeutic situation. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of active agentcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

Administration may be in single or multiple doses. Multiple doses may beadministered via the same or different routes and to the same ordifferent locations. Alternatively, doses can be via a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency may vary depending on the half-life of theinhibitory agent in the patient and the duration of treatment desired.

Agents, formulations or pharmaceutical compositions for use in theinvention may be co-administered with one or other more othertherapeutic agents. Combined administration of two or more agents may beachieved in a number of different ways. Both may be administeredtogether in a single composition, or they may be administered inseparate compositions as part of a combined therapy. For example, theone may be administered before, after or concurrently with the other.

Therapeutic Indications

Inhibitors of gremlin-1 activity according to the present invention areprovided for the treatment of a bone fracture or bone defect. A bonefracture is a break or crack in bone tissue and may be the result of atraumatic injury, such as a fall or impact, but can also occur as aresult of diseases that affect bone integrity. A bone defect is a lossof bone, due to trauma or disease.

The fracture may be a fracture of any bone in the body.

The bone defect may be a bone defect in any bone of the body.

In one embodiment, the bone fracture is a delayed-union or non-unionfracture. A delayed-union fracture is defined as a fracture which failsto reach union within 6-months post-fracture. A non-union fracture isdefined as incomplete healing within 9 months, combined with a lack ofradiological characteristics associated with fracture healing beingobserved over three consecutive months. (Einhorn et al; 2014; Buza etal; 2016). Examples of fractures that are that are prone todelayed-union or non-union development include tibia, distal radius,femoral neck and scaphoid.

In one embodiment, the bone fracture or bone defect occurs as a resultof a disease that affects bone integrity. Examples of diseases thataffect bone integrity include but are not limited to osteoporosis,osteogenesis imperfecta, diabetes, Paget's disease of bone, rheumatoidarthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer(e.g. osteosarcoma, Ewing's sarcoma and chondrosarcoma), cancers thatmetastasise to the bone (e.g. breast cancer, prostate cancer and lungcancer), diffuse idiopathic skeletal hyperostosis, osteomyelitis, renaldisease, Duchenne muscular dystrophy and thalassemia major.

FIGURES

FIG. 1 shows percentage restoration of signal for the immunisationderived antibodies in the HEK-ID1 reporter gene assay.

FIG. 2 shows percentage restoration of signal for library derivedantibodies in the HEK-ID1 reporter gene assay.

FIG. 3 shows results for the HEK-ID1 reporter gene assay with titrationsof human Gremlin (FIG. 3A) and mouse Gremlin (FIG. 3B) and the effect ofantibody 7326 (shown as antibody PB376) in restoring signalling of BMP.

FIG. 4 shows a structural model of the Gremlin-Fab complex, with thepossible BMP binding regions and the Fab epitope highlighted.

FIG. 5 shows examination of the area devoid of callus/bone tissue duringfracture repair in acquired X-Ray images. The area within the defect,which was devoid of tissue, was quantified using definiens imageanalysis and subsequently compared between control and anti-gremlin 1treated group. Results are presented as the mean±SD of 10 rats/group.*P<0.05; **P<0.01; ***P<0.001 as measured by Mann-Whitney U test.

FIG. 6 shows examination of LMB (low mineral bone; newly formed bone)and HMB (high mineral bone; mature bone) within a 3 mm femoral bonedefect. Panel A: 3D μCt analysis of femoral bone defect region to detectnewly formed bone or mature bone. Percentage of bone volume/tissuevolume was measured and compared all subjects (total) between controlvs. treatments with anti-gremlin 1. Comparisons between control versusanti-gremlin 1 treated group in animals separated as low responders (LR-incomplete bridging) and high responders (HR complete bridging) werealso performed. Results are presented as the mean ±SD. *P<0.05;**P<0.01; ***P<0.001 as measured by Mann-Whitney U test. Panel B:representative μCt illustrating the 3D bone volume renderings of LR andHR groups in control and after anti-gremlin 1 treatment.

FIG. 7 shows histomorphometric analysis of femoral bone defect.Percentage of bone volume/tissue volume (BV/TV ((Yip)), trabecularnumber (Tb.N) and trabecular separation (Tb.Sp) was compared betweencontrol versus anti-gremlin 1 treated group. Results are presented asthe mean±SD of 10 rats/group. *P<0.05; **P<0.01; ***P<0.001 as measuredby Mann-Whitney U test.

FIG. 8 shows correlation of 3D μCT analysis and 2D histomorphometryanalysis of total BV/TV %. Correlations were performed in both groups onLMB (low mineral bone; newly formed bone) and HMB (high mineral bone;mature bone) within a 3 mm femoral bone defect and compared to 2Dhistomorphometry score data (n=20). The Pearson's score indicatessignificant correlation of BV/TV % between 3D μCT analysis and 2Dhistomorphometry analysis.

The following Examples illustrate the invention.

EXAMPLES Example 1—Protein Expression, Purification, Refolding andStructure Determination

Protein Expression and Inclusion Body Preparation

A truncated human Gremlin-1 coding sequence (SEQ ID NO: 20), optimisedfor expression in E. coli, was cloned into a modified pET32a vector(Merck Millipore) using BamHI/Xhol, generating a vector encoding theGremlin sequence with an N-terminal 6His-TEV tag (pET-hGremlin1).

Expressed sequence:

MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD;SEQ ID NO: 2 (with non-Gremlin residues of the 6His-TEV tag shown initalics). Sequence numbering based on UniProt 060565 & SEQ ID NO: 1.

The pET-hGremlin1 plasmid DNA was used to transform BL21(DE3) cells. Asingle ampicillin resistant colony was picked from a LB/Amp agar plateand used to inoculate a 100 ml starter culture of LB/Amp. After shaking(200 rpm) for 16 hr at 37° C., 25 ml of the starter culture was used toinoculate 500 mL of 2×TY/Amp media. The culture was shaken (250 rpm) at37° C. until an OD₆₀₀ of 3 was achieved. Subsequently, the culture wassupplemented with 20 mL of a MOPS+glycerol feed mix (1M MOPS pH 7.4, 40%glycerol, 0.5% MgSO₄, 0.42% MgCl₂), induced with 300 μM IPTG and furtherincubated at 17° C., 180 rpm for 16 hours. Cells were harvested in acentrifuge (4,000 g for 20 minutes at 4° C.).

Cell pellets were resuspended in Lysis Buffer (PBS pH 7.4, 0.35 mg/mllysozyme, 10 μg/ml DNase and 3 mM MgCl₂) at 4° C. and the insolublefraction was harvested by centrifugation at 3,500 g for 30 minutes at 4°C. Pelleted inclusion bodies were washed three times by resuspending inwash buffer (50 mM Tris, 500 mM NaCl, 0.5% Triton X-100, pH 8.0),followed by centrifugation at 21,000 g for 15 minutes. An additional twowashes were performed using wash buffer without Triton X-100.

Solubilisation

Inclusion bodies were resuspended in denaturing buffer (8 M Urea, 100 mMTris, 1 mM EDTA, 10 mM Na₂S₄O₆ and 100 mM Na₂SO₃, pH 8.5), stirred for16 hrs at room-temperature and clarified by centrifugation at 21,000 gfor 15 minutes.

Pre-Refolding Purification

The solubilized inclusion bodies were loaded onto a Sephacryl S-20026/60 column (120 mL) equilibrated in 8 M Urea, 50 mM MES, 200 mM NaCl,1 mM EDTA, pH 6.0. Fractions containing Gremlin-1 protein were dilutedwith 6 M Urea, 20 mM MES, pH 6.0 and loaded onto HiTrap SP HP cationexchange columns and eluted with a 1 M NaCl gradient over 10 columnvolumes (10 CVs). Fractions containing purified, denatured hGremlin-1protein were pooled.

Refolding

Denatured purified Gremlin-1 protein was added drop-wise to re-foldingbuffer (50 mM Tris, pH 8.5, 150 mM NaCl, 5 mM GSH and 5 mM GSSG, 0.5 mMCysteine, 5 mM EDTA, 0.5 M Arginine) to a final concentration of 0.1mg/ml and incubated at 4° C. with constant stirring for 5 days. After 5days the Gremlin-1 protein was dialysed against 20 mM HEPES, 100 mMNaCl, pH 7.5.

Following dialysis protein was applied to heparin HiTrap column andeluted using a gradient of 0-100% heparin elution buffer (20 mM HEPES, 1M NaCl, pH 7.5) over 20 CV. Correctly folded protein eluted at 1 M NaClwhereas any misfolded protein eluted at lower salt concentrations.

Protein eluting at 1 M NaCl was concentrated and purified further on aS75 26/60 column equilibrated with 20 mM Hepes, pH 7.5, 1 M NaCl.

Protein was characterised by SDS PAGE (shift in gel), demonstrated tohave the expected molecular weight and correct arrangement of disulphidebonds using liquid chromatography mass spectrometry (LC-MS) and to beactive in a cell assay (ID1 reporter assay).

Gremlin 1 Structure Determination

Gremlin 1 protein crystals were grown using the hanging-drop method bymixing a solution of Gremlin 1 at 6.6 mg/ml and 0.1 M citric acid at pH4, 1 M lithium chloride and 27% polyethylene glycol (PEG) 6000 in a 1:1ratio. Before data collection, crystals were cryo-protected by adding20% glycerol to the crystallization buffer. Diffraction data werecollected at the Diamond Light Source and were processed using XDS(Kabsch, Wolfgang (2010) Acta Crystallographica Section D 66, 125-132).Diffraction data statistics are summarized in the table below:

TABLE 2 Diffraction data statistics Diffraction Statistics Wavelength(Å) 0.97949 Space group C2 Cell dimensions a = 84.55 Å, b = 107.22 A, c= 77.09 Å; α = 90.00°, β = 120.43°, γ = 90.00° Resolution range* (Å)26.19-2.72 (2.79-2.72) Completeness (%) 98.5 (99.0) Multiplicity 3.4(3.4) I/sigma 9.6 (2.0) Rmerge 0.095 (0.622) Refinement StatisticsResolution Range (Å) 26.19-2.72 R_(cryst) 0.24 R_(free) 0.29 R.m.s.d.bonds (Å)** 0.013 R.m.s.d. angles (°) 1.782 *values in parenthesiscorrespond to the highest resolution shell **r.m.s.d root mean squaredeviation

Gremlin-1 structure was solved by molecular replacement using Phaser(McCoy et al, J Appl Cryst (2007), 40, 658-674) and a Gremlin-1 modelavailable from proprietary Gremlin-1/Fab complex coordinates. Theresultant model of Gremlin-1 contained four copies of Gremlin 1 monomerorganised as two dimers. Model corrections were made with Coot (Emsleyet al Acta Crystallographica Section D: Biological Crystallography 66(4), 486-501) and coordinates were refined using Refmac (Murshudov et alREFMAC5 for the refinement of macromolecular crystal structures. ActaCrystallographica Section D: Biological Crystallography. 2011;67(Pt4):355-367). Final coordinates were validated with Molprobity (Chen etal. (2010) MolProbity: all-atom structure validation for macromolecularcrystallography. Acta Crystallographica D66:12-21). A summary of modelrefinement statistics is shown in Table 2 above.

Example 2—BMP Binding Residues on Gremlin-1

As discussed above, Gremlin-1 belongs to the bone morphogenic protein(BMP) antagonist protein family within a sub-group known as the DANfamily. Within the DAN family, Gremlin-1 shares greatest homology withGremlin-2 (PRDC).

The 2.7 A human Gremlin-1 structure resolved in Example 1 shares manyfeatures in common with the published mouse Gremlin-2 structure (Nolanet al (2013), Structure, 21, 1417-1429). The overall fold is verysimilar, with two copies of Gremlin-1 forming an antiparallel,non-covalent dimer, arranged in an arch. Each monomer adopts thecharacteristic finger-wrist-finger arrangement with a cystine-knot motiftowards the wrist end, opposite the fingers. Sequence identity betweenthe proteins is 52% rising to 67% within the sequence visible in the twostructures. The most highly conserved region lies in the extensive dimerinterface where all the key contact residues are 100% conserved.

Residues involved in BMP's 2, 4 & 7 binding to mouse Gremlin-2 (PRDC)and DAN (NBL1) have been identified using mutagenesis (Nolan et al(2013), Structure, 21, 1417-1429 and Nolan et al (2014) J. Biol. Chem.290, 4759-4771). The predicted BMP binding epitope encompasses ahydrophobic patch spanning across both monomers on the convex surface ofthe dimer. Six residues were identified by mutagenesis; Trp72, Phe96,Tyr98, Phe104, Tyr105 & Phe117 and are 100% conserved in human Gremlin-1(numbering based on the mouse Gremlin-2 sequence). The degree ofhomology extends to the positioning of the side chains which adopt anidentical conformation in both proteins.

The amino acid numbering used in the Gremlin PDB file matches thenumbering in the published mouse Gremlin-2 structure based on astructural alignment. This enables like for like comparison of aminoacids when describing the structures. However, for clarity the keyresidues identified as playing a role in BMP binding are shown belowwith numbering based on the PDB file and UniProt file of SEQ ID NO: 1 inbrackets:

Trp72(93), Phe96(117), Tyr98(119), Phe104(125), Tyr105(126) &Phe117(138).

In both mouse Gremlin-2 and human Gremlin-1 the hydrophobic BMP bindingepitope is partially buried by an alpha helix formed by the N-terminalresidues of each protein. A model of BMP binding has been proposedwhereby the N-terminus can flex, exposing the full BMP binding interface(Nolan et al (2013), Structure, 21, 1417-1429). In the present analysis,the N-terminal residues were removed from the human Gremlin-1 and mouseGremlin-2 structures before rendering a surface to reveal the similarityof the BMP binding faces on each protein.

The literature only describes mutagenesis of six resides that have aneffect on BMP binding. It is possible that the actual BMP epitope coversa larger surface area, encompassing neighbouring amino acids. Byhighlighting all residues, within 6 Å of those mutated, on the surfaceof Gremlin-1, a larger region of Gremlin-1 is revealed that couldpotentially be targeted by a therapeutic. This more extensive regionencompasses the following amino acids of human Gremlin-1:

-   -   Asp92-Leu99    -   Arg116-His130    -   Ser137-Ser142    -   Cys176-Cys178    -   (Numbering based on SEQ ID NO: 1)

By combining published information with the crystal structureinformation of human Gremlin-1, regions of human Gremlin-1 that offerthemselves as a potential route for therapeutic intervention blockingits interaction with BMP's have been identified.

Example 3—Hek Id1 Reporter Gene Assay

Background

The Hek Id1 reporter gene assay uses Clone 12 Hek293-Id1 reporter cells.This cell line was stably transfected with Id1 transcription factor. Id1is a transcription factor in the BMP signalling pathway. Gremlin isknown to bind BMPs prevent binding to their receptors reducing theluciferase signal from the reporter gene. Therefore, using this reporterassay, it is possible to screen anti-Gremlin antibodies and see if thereare any that block the interaction of Gremlin with BMPs. A restorationof the luciferase signal is seen in these cells if there is a blockingof this interaction.

Method

Clone 12 cells were cultured in DMEM containing 10% FCS, 1×L-Glutamine &1×NEAA. Cells are also grown in the presence of Hygromycin B (200 μg/ml)to ensure cells do not lose Idi gene expression. Cells were assayed inDMEM containing 0.5% FCS, 1×L-Glutamine & 1×NEAA. Hygromycin B is notneeded for the short time that the cells are in the assay.

The cells were washed in PBS, lifted off using cell dissociation buffer,spun and counted before being seeded at 5×104/well in 70 μl (Density of7.14×10⁵/ml). Plates used were white, opaque Poly-D-Lysine coated96-well sterile. Cells go in incubator for about 3-4 hours to settledown. BMP heterodimers were reconstituted to 200 μg/ml in 4 mM HCL. BMPwas diluted to 10 μg/ml in assay media using a glass vial to give a newworking stock.

In a polypropylene plate, Gremlin-1 was diluted 1:2 for an 8 point doseresponse curve with a top final dose of 1 μg/ml.

An additional volume of 20 μl media was added per well and plates wereincubated at 37° C. for 45 mins.

BMP prepared at 100× was added to all wells except wells containingcells only. All wells are made up to 60 μl with assay medium andincubated for a further 45 mins at 37° C.

Post incubation, 30 μl of sample was transferred per well of assay plateand incubated for 20-24 hours before measuring luminescence signal.

Cell Steady Glo was thawed in advance at room temperature. Assay plateswere cooled to room temperature for about 10-15 mins before adding thereagent. Luciferase signal was detected by addition of cell steady gloreagent (100 μl) for 20 minutes on shaker at room temperature andmeasuring luminescence using cell titre glo protocol on Synergy 2.

The maximum signal was generated from wells containing BMP and theminimum signal was generated from the wells containing cells only.

Results

Gremlin-1 full length and truncated forms were tested in the Hek-Id1reporter gene assay to confirm the blocking activity against BMP4/7.

The percentage of inhibition from dose response assays was calculatedbased on the maximum and minimum signals in the assay and the datafitted using 4 parameter logistical fit. The IC₅₀ was calculated basedon the inflexion point of the curve.

TABLE 3 Potency results for full length Gremlin-1 and truncatedGremlin-1 in the Hek-Id1 reporter gene assay. Hek-Id1 Reporter Geometric95% CI (or range gene assay N mean (nM) where N = <4) Gremlin 1 Full 21.6 1.3-1.9 length Gremlin 1 2 1.7 1.1-2.5 truncated

Conclusion

Gremlin 1 was able to inhibit the BMP 4/7 signalling in the Hek-Id1reporter gene assay.

Example 4—Production of Anti-Gremlin-1 Antibodies

Anti-Gremlin-1 antibodies were derived by immunisation using purifiedgremlin-1 as described in Example 1, and by library panning. The librarywas generated in-house as a naive human library with the V-regionsamplified from blood donations.

Immunisation yielded 26 distinct antibodies binding Gremlin-1 from thefirst round of immunisation. These antibodies were scaled up andpurified for testing in screening assays.

25 human and mouse cross-reactive antibodies from the library werepanned using recombinant human Gremlin from R&D Systems. 10 antibodieswere selected for scale up and purified as scFvs for testing in thescreening assays.

Example 5—Screening of Anti-Gremlin-1 Antibodies

Antibodies were screened using the Hek-Id1 reporter gene assay describedin Example 3 and by measuring SMAD phosphorylation. SMAD1, 5 and 8 arephosphorylated upon BMP signalling. Inhibitors of Gremlin-1 thereforeincrease SMAD phosphorylation.

SMAD phosphorylation assays were conducted on A549 cells or on humanlung fibroblasts. Phosphorylation levels were determined using MSD.

Results

In the Hek-Id1 reporter gene assay, there were no apparent hits with theimmunisation derived antibodies (with a 10 fold excess of antibodytested against a BMP4/7 heterodimer). Results are shown in FIG. 1 .

In contrast, a number of library derived antibodies were capable ofrestoring signal in the Hek-Id1 reporter gene assay (50-fold excess ofantibodies with a 50% gremlin dose) (FIG. 2 ). Of these, Ab2416 andAb2417 contained high levels of endotoxin. Ab7326 maintained blockingability at a 10-fold excess and 80% inhibition Gremlin-1 concentration.

Additional results are presented in FIGS. 3A (human gremlin) and 3B(mouse Gremlin). These Figures show titrations of Ab7326 (labelled asPB376) up to 15 nM. Ab7326 was shown to restore signalling of BMP whenblocked by either human (IC₅₀ of 1.3 nM) or mouse (IC₅₀ of 0.2 nMGremlin). The antibody functions both as a human and mouse IgG1.

Sequences of the mouse and human full length IgG1 are presented below.In order to synthesise the mouse and human full length IgG1 proteins,the Ab7326 variable regions derived from the library were re-cloned intovectors comprising the appropriate antibody constant domains.

Because Ab7326 came from a naïve human library, where Abs are cloned asscFvs, in order to re-clone the 7326 variable regions as full length Absor Fabs, it was necessary to PCR amplify the VH and VK using pools ofprimers/degenerate primers. The amplified PCR products were thendigested and cloned simultaneously into mouse and human vectors. As theVH and VK were amplified by pools of primers/degenerate primers, twovariant forms of the products were obtained, differing by a single aminoacid residue derived from subtly different primers annealing during thePCR process.

The two variant forms of heavy chain variable region differed by asingle amino acid at position 6, and the two variant forms of the lightchain variable region differed by a single amino acid at position 7, asshown below:

-   -   Heavy chain variable region variant 1 has glutamic acid (E) at        position 6.    -   Heavy chain variable region variant 2 has glutamine (Q) at        position 6.    -   Light chain variable region variant 1 has serine (S) at position        7.    -   Light chain variable region variant 2 has threonine (T) at        position 7.

Mouse full length IgG1 - heavy chain variant 1 (SEQ ID NO: 14)QVQLVESGAE VKKPGATVKI SCKVSGYTFT  DYYMHWVQQAPGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAYMELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLVTVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEPVTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWPSETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSSVFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFVDDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEFKCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEQMAKDKVSLTCMI TDFFPEDITV EWQWNGQPAE NYKNTQPIMDTDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGKMouse full length IgG1 - light chain variant 1 (SEQ ID NO: 15)DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLAWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTINSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTDAAPTVSIFPPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSERQNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNECMouse full length IgG1 - heavy chain variant 2 (SEQ ID NO: 28)QVQLVQSGAE VKKPGATVKI SCKVSGYTFT DYYMHWVQQAPGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAYMELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLVTVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEPVTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWPSETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSSVFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFVDDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEFKCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEQMAKDKVSLTCMI TDFFPEDITV EWQWNGQPAE NYKNTQPIMDTDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGKMouse full length IgG1 - light chain variant 2 (SEQ ID NO: 29)DIVMTQTPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLAWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTINSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTDAAPTVSIFPPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSERQNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNECHuman full length IgG1 - heavy chain variant 1 (SEQ ID NO: 30)QVQLVESGAE VKKPGATVKI SCKVSGYTFT  DYYMHWVQQAPGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAYMELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLVTVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEPVTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSLGTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPELLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEVKFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPPSRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKTTPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKHuman full length IgG1 - light chain variant 1 (SEQ ID NO: 31)DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLAWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTINSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGECHuman full length IgG1 - heavy chain variant 2 (SEQ ID NO: 16)QVQLVQSGAE VKKPGATVKI SCKVSGYTFT  DYYMHWVQQAPGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAYMELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLVTVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEPVTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSLGTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPELLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEVKFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPPSRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKTTPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKHuman full length IgG1 - light chain variant 2 (SEQ ID NO: 17)DIVMTQTPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLAWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTINSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Antibody CDRs were determined using the Kabat method (highlighted inbold in the above sequences). Additional HCDR1 residues using theChothia definition are in italics. Constant region sequences areunderlined.

Restoration of p-SMAD signalling with anti-Gremlin 1 antibodies is shownin Table 4 below.

TABLE 4 Restoration of p-SMAD signalling 2417 2418 2419 2481 2482 24832484 7326 8427 BMP 2 109.1% +/− 58.2% +/− 32.6% +/− 40.4% +/− 35.3% +/−43.1% +/− 104.0% +/− 107.2% +/− 51.3% +/− 50 ng/ml 2.8% 1.9% 1.4% 0.6%0.8% 2.1% 2.7% 3.5% 1.4% BMP 4 109.6% +/− 71.3% +/− 31.7% +/− 60.1% +/−54.4% +/− 72.5% +/− 105.2% +/− 110.0% +/− 78.2% +/− 25 ng/ml 3.0% 3.1%1.2% 2.2% 1.3% 2.1% 3.3% 3.8% 2.5% BMP 7 111.5% +/− 99.5% +/− 53.8% +/−64.4% +/− 52.3% +/− 66.2% +/− 105.2% +/− 108.0% +/− 72.6% +/− 200 ng/ml3.8% 3.2% 3.4% 1.3% 1.1% 1.2% 4.3% 3.2% 2.5% BMP-2/7 119.3% +/− 78.6%+/− 50.8% +/− 53.7% +/− 47.6% +/− 56.1% +/− 120.4% +/− 128.5% +/− 62.8%+/− 50 ng/ml 2.6% 3.6% 2.7% 3.1% 1.5% 2.5% 4.4% 2.9% 2.5% BMP4/7 113.7%+/− 78.0% +/− 61.4% +/− 48.3% +/− 41.7% +/− 50.8% +/− 112.4% +/− 127.0%+/− 63.3% +/− 50 ng/ml 3.1% 4.0% 4.0% 2.1% 1.7% 1.7% 2.5% 3.1% 2.1%

Results are shown as a percentage of SMAD phosphorylation by BMP alone(control BMP). Experiments were performed using lung fibroblasts fromidiopathic pulmonary fibrosis patients. rhGremlin-1 and theanti-Gremlin-1 antibodies were preincubated for 45 minutes at roomtemperature. rhGremlin-1 and the anti-Gremlin-1 antibodies were thenadded with BMP to the cells for 30 minutes.

Table 5 then shows further results in the SMAD phosphorylation assay,where displacement of BMP-2 or BMP4/7 from Gremlin 1-BMP complexes byanti-Gremlin-1 antibodies was investigated. Experiments were againperformed using lung fibroblasts from idiopathic pulmonary fibrosispatients. rhBMP-2 or rhBMP 4/7 were preincubated with rhGremlin-1 for 1hour at room temperature. The BMP-2-or BMP4/7-Gremlin-1 complexes wereincubated with different concentrations of the anti-Gremlin-1 antibodiesovernight at 4° C. Antibody concentrations represent the finalconcentration on the plate.

TABLE 5 Displacement of BMP-2 or BMP4/7 from Gremlin 1-BMP complexes byanti-Gremlin-1 antibodies 81.3 μg/ml 40.6 μg/ml 20.3 μg/ml 10.2 μg/ml5.1 μg/ml 2.55 μg/ml 1.27 μg/ml 0.63 μg/ml 2484 BMP 2 100.3% +/− 98.8%+/− 97.0% +/− 93.5% +/− 86.4% +/− 79.9% +/− 66.5% +/− 54.8% +/− 50 ng/ml3.5% 2.7% 2.9% 2.6% 2.0% 1.9% 2.8% 0.3% 2484 BMP4/7 136.4% +/− 133.2%+/− 121.4% +/− 108.1% +/− 86.6% +/− 74.7% +/− 65.8% +/− 60.7% +/− 50ng/ml 4.2% 1.0% 1.4% 4.9% 4.4% 2.2% 0.6% 1.5% 7326 BMP 2 103.7% +/−101.5% +/− 99.4% +/− 103.8% +/− 100.3% +/− 103.2% +/− 102.8% +/− 97.0%+/− 50 ng/ml 1.1% 2.4% 3.8% 2.4% 2.2% 4.3% 2.8% 2.9% 7326 BMP4/7 133.7%+/− 132.3% +/− 130.3% +/− 125.6% +/− 121.4% +/− 120.9% +/− 111.1% +/−102.0% +/− 50 ng/ml 0.8% 1.8% 4.2% 10.0% 4.2% 3.3% 2.3% 4.5%

The results shown in Table 5 demonstrate that Ab7326 can displacealready complexed BMP-2 or BMP4/7 from Gremlin 1-BMP complexes. Ab7326can achieve this displacement at much lower concentrations that thecomparison antibody 2484. This provides evidence that Ab7326 is anallosteric inhibitor, consistent with our finding that the binding sitefor Ab7326 is distal from the known BMP binding regions on gremlin-1.Thus Ab7326 is able to access the allosteric binding site even when BMPis complexed to gremlin-1, resulting in significantly improvedinhibition of gremlin activity.

Example 6—Obtaining the crystal structure of Gremlin-1 in complex withthe 7326 Fab

The crystal structure of human Gremlin-1 in complex with Ab7326 Fab wassolved at a resolution of 2.1 Å. Fab sequences are shown below:

Heavy chain: SEQ ID NO: 18QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Light chain: SEQ ID NO: 19DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

The CCP4 software NCONT was then used to identify all contacts at 4 Åbetween Gremlin-1 and the Fab. The following residues were identified:Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 andGIn175 (numbering based on the UniProt Sequence of SEQ ID NO: 1(numbered as Ile110, Lys126, Lys127, Phe128, Thr129, Thr130, Arg148,Lys153 and GIn154 in the structure file which matches the numbering ofmouse Gremlin-2).

FIG. 4 shows structural models of the Gremlin-Fab complex, with the Fabepitope residues shown relative to the BMP binding regions.

Ab7326 is an inhibitory antibody which acts allosterically, i.e. itbinds away from the BMP binding regions.

Example 7—Affinity Measurements for Binding of Anti-Gremlin-1 AntibodyAb7326 to Gremlin-1.

Method

The affinity of anti-Gremlin mlgG for human Gremlin 1 was determined bybiamolecular interaction analysis using surface plasmon resonance (SPR)technology on a Biacore T200 system, GE Healthcare Bio-Sciences AB.Anti-Gremlin mlgG was captured by an immobilised anti-mouse Fc surfaceand Gremlin 1 was titrated over the captured mlgG. The capture ligand(affinipure F(ab′)₂ fragment of goat anti-mouse IgG, Fc fragmentspecific, 115-006-071, Jackson ImmunoResearch Inc.) was immobilised at50 μg/ml in 10 mM NaAc, pH5.0 on flow cell 2 of a CM4 Sensor Chip viaamine coupling chemistry, using 600 s activation and deactivationinjections, to a level of ˜1600 response units (RU). HBS-EP+ buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) wasused as the running buffer with a flow rate of 10 μl/min. A referencesurface was prepared on flow cell 1 by activating and deactivating thesurface as for flow cell 2 but omitting the capture ligand.

The assay buffer was HBS-EP+ plus an extra 150 mM NaCl to give a finalNaCl concentration of 300 mM plus 1% CMD40. A 60 s injection ofanti-Gremlin mlgG (at 5 μg/ml in running buffer) was passed over flowcells 1 and 2 to give a capture level of approximately 100 RU on theimmobilised anti-mouse IgG, Fc surface. Recombinant human Gremlin 1 wastitrated in running buffer from 5 nM (using 2-fold dilutions) andinjected over flow cells 1 and 2 at a flow rate of 30 μl/min for 3 minfollowed by a 5 min dissociation phase. A buffer only control was alsoincluded. The surface was regenerated at a flow rate of 10 μl/min by a60 s injection of 50 mM HCl, a 30 s injection of 5 mM NaOH and a 30 sinjection of 50 mM HCl.

The kinetic data was determined using Biacore T200 evaluation software.

The affinity measurements were made at 25° C.

Results

Binding affinity, taken as the average K_(D) value for 5 determinations,was found to be below 100 pM.

Example 8. Inhibition of Gremlin-1 Activity Accelerates Healing andBridging in an in Vivo Model of Bone Fracture Repair

8.1. Materials and Methods

Rat Fracture Model and Drug Administration

Long bone segmental defect models have been widely used for the researchof bone healing and regeneration (Sato et al; 2014). In the presentstudy, a 3 mm femoral defect was created in 10-week old male rats andstabilised using an 8-hole PEEK plate (RIS. 602.105, RISystem,Switzerland). The plate was fixed to the bone with a forceps in themiddle of the diaphysis, before the bone was drilled and fixed withscrews. A 3 mm fracture gap was created using a 0.44 mm Gigly saw. Thedefect size/consistency/fixation was quality controlled by X-Ray imagingusing Faxitron (MX-20-DCS, Faxitron Bioptics LLC, USA), this time pointwas defined as Day 0.

Weekly dosing was commenced on Day 1 for a period of 8 weeks as outlinedin Table 1. X-ray images were subsequently acquired during the in-lifephase of the study at day 11, 25, 39 and 57 in order to assess thecallus formation and the progress of healing. Definiens image analysiswas utilized to quantify the area of the defect that was devoid of bonetissue in the captured X-Ray images.

TABLE 6 Treatment Groups. Animal Dosing Group Number Treatment DoseRegimen Time 1 10 Vehicle Vehicle: Once/week Total of 1 ml, s.c. 57 days2 10 Anti- 30 mg/kg, Once/week Gremlin-1 1 ml, s.c.

Micro-CT Analysis of Fracture Healing

Femora (fractured side) were scanned at 17.2 μm resolution usingmicro-CT (SkyScan 1076). A region of approximately 15 mm of the calluswith the fracture in the centre was acquired. The scans werereconstructed using the Skyscan NRECON software (1.7.10) and then thereconstructed slices were further segmented to exclude fixator pins at a3 mm defined region calculated from the mid-point of the femoralfracture defect.

Histomorphometric analysis of fracture callus in 3D was performed bySkyScan software (v. 1.13.1). The mid-point within the 3mm femoralfracture defect was determined and slices 1.5 mm distal and proximal tothe mid-point were segmented for each limb measured. Subsequently, thebinarization of the reconstructed datasets and segmentation wereperformed following two defined thresholds, one to delineate the lowmineralized callus (thus quantifying newly formed bone) and the otherone to define mature bone. Further segmentation of these data wascarried out on femora of animals classified as low responders based onsatisfying the criteria of incomplete bridging of the femoral defect orhigh responders exhibiting bridging of the fracture site.

Histomorphometry Analysis of Fracture

Femora were fixed in 10% neutral-buffered formalin for 24 h, dehydratedand embedded in methyl methacrylate (MMA) at low temperature.50-μm-thick sections were stained with Toluidine Blue to quantify thebone elements of the healing gap defect. Histomorphometric parameterswere measured on the trabecular bone of the fracture defect site.Measurements were performed through image analysis.

Statistical Analysis

The results were presented as mean values±SD. Statistical analysis wasperformed using a two-tailed Mann Whitney U test with GraphPad Prismsoftware unless otherwise stated.

8.2 Results

Analysis of X-Ray images obtained during the in-life phase of the studyindicated that the anti-gremlin-1 antibody accelerated fracture healingwith the control and treated groups significantly diverging after 25days (P<0.05). This effect was apparent for the remainder of the study(FIG. 5 ).

Micro-CT analysis of terminal samples revealed that treatment withanti-gremlin-1 antibody (30 mg/kg/once weekly) led to an increase innewly formed bone within the fracture callus site (P=0.06).

The incidence of fracture non-union in this model is approximately 60%with no intervention (Sato et al; 2014). To test whether gremlin-1inhibition reduced the incidence of non-union development, the animalswere classified as low responders (LR) and high responders (HR).Gremlin-1 inhibition resulted in a significant increase in thepercentage of bone volume/tissue volume (BV/TV %) within LMB (lowmineral bone; newly formed bone) (P<0.01) and HMB (high mineral bone;mature bone) (P<0.01) in the low responder group compared to controls,thus indicating progressive repair of the cohort likely to formnon-union.

Additionally, there was a trend (non-significant) towards increased LMBand HMB BV/TV % in the high responder group in response toanti-gremlin-1 treatment (FIG. 6A, representative images of LR and HRare shown in FIG. 6B).

Two-dimensional histomorphometric analysis of bone parameters wasperformed on histological sections of the fracture site (FIG. 7 ).Treatment with anti-gremlin-1 antibody significantly increasedpercentage of bone volume/tissue volume (BV/TV %) (P<0.05) compared tocontrol. Anti-gremlin-1 significantly increased trabecular number (Tb.N)(P<0.001) and significantly decreased trabecular separation (Tb.Sp)(P<0.01) indicating increased trabecular bone due to treatment withanti-gremlin-1.

Correlations were performed between two-dimensional histomorphometricanalysis and three-dimensional μCT analysis by comparing the LMB and HMBgroups segmented in μCt analysis and the two-dimensionalhistomorphometry analysis of fracture sections (FIG. 8 ). Comparisonsmeasured by Pearson's correlation revealed a positive and significantcorrelation between histomorphometry and μCT analysis in the LMB(P<0.0001) and HMB groups (P<0.0001) thus validating the data from eachdata-set.

8.3. Conclusion

Inhibition of gremlin-1 activity using a neutralising anti-gremlin-1antibody resulted in accelerated fracture repair, with significantdifferences between control and treated groups evident after 25 days (3doses of antibody). Additionally, terminal analysis of the fracture siteindicated the enhanced formation of bone tissue in the low responderanimals, which otherwise would likely form non-union. Therefore,inhibition of gremlin-1 activity is a promising therapy for theprevention or treatment of non-union fractures and may be of particularvalue for the treatment of fractures that are prone to non-uniondevelopment, for example, tibia, distal radius, femoral neck andscaphoid.

SEQUENCE LISTING (Human Gremlin-1; Uniprot ID: O60565) SEQ ID NO: 1MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDL D(Human truncated Gremlin-1 used in crystallography with N-terminal tag)SEQ ID NO: 2MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD (Ab7326 HCDR1 combined Kabat & Chothia) SEQ ID NO: 3GYTFTDYYMH (Ab7326 HCDR1 Kabat) SEQ ID NO: 4 DYYMH (Ab7326 HCDR2 Kabat)SEQ ID NO: 5 LVDPEDGETIYAEKFQG (Ab7326 HCDR3 Kabat) SEQ ID NO: 6DARGSGSYYPNHFDY (Ab7326 LCDR1 Kabat) SEQ ID NO: 7 KSSQSVLYSSNNKNYLA(Ab7326 LCDR2 Kabat) SEQ ID NO: 8 WASTRES (Ab7326 LCDR3 Kabat)SEQ ID NO: 9 QQYYDTPT (Ab7326 Heavy chain variable region variant 1)SEQ ID NO: 10QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTV SS(Ab7326 Light chain variable region variant 1) SEQ ID NO: 11DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK(Ab7326 Heavy chain variable region variant 2) SEQ ID NO: 12QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTV SS(Ab7326 Light chain variable region variant 2) SEQ ID NO: 13DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK(Mouse full length IgG1 heavy chain variant 1) SEQ ID NO: 14QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (Mouse full length IgG1 light chain variant 1)SEQ ID NO: 15DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(Human full length IgG1 heavy chain variant 2) SEQ ID NO: 16QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Human full length IgG1 light chain variant 2) SEQ ID NO: 17DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fab heavy chain variant 1)SEQ ID NO: 18QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (Fab light chain variant 1)SEQ ID NO: 19DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Human truncated Gremlin-1 used incrystallography without N-terminal tag) SEQ ID NO: 20AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD(Mature Gremlin-1 sequence of SEQ ID NO: 1lacking the signal peptide of amino acids 1-21) SEQ ID NO: 21KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD(Human IgG4P heavy chain variant 1) SEQ ID NO: 22QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Human IgG4P light chain variant 1)SEQ ID NO: 23DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Human IgG1 heavy chain DNA variant 1) SEQ ID NO: 24caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagatctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacctgggaagggccttgagtggatgggtctggtcgatcccgaggacggcgaaactatctacgccgagaagttccagggtcgcgtcaccatcaccgccgacacttccaccgacaccgcgtacatggagctgtccagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagctactacccgaaccacttcgactactggggacagggcactctcgtgactgtctcgagcgcttctacaaagggcccctccgtgttcccgctcgctccatcatcgaagtctaccagcggaggcactgcggctctcggttgcctcgtgaaggactacttcccggagccggtgaccgtgtcgtggaacagcggagccctgaccagcggggtgcacacctttccggccgtcttgcagtcaagcggcctttactccctgtcatcagtggtgactgtcccgtccagctcattgggaacccaaacctacatctgcaatgtgaatcacaaacctagcaacaccaaggttgacaagaaagtcgagcccaaatcgtgtgacaagactcacacttgtccgccgtgcccggcacccgaactgctgggaggtcccagcgtctttctgttccctccaaagccgaaagacacgctgatgatctcccgcaccccggaggtcacttgcgtggtcgtggacgtgtcacatgaggacccagaggtgaagttcaattggtacgtggatggcgtcgaagtccacaatgccaaaactaagcccagagaagaacagtacaattcgacctaccgcgtcgtgtccgtgctcacggtgttgcatcaggattggctgaacgggaaggaatacaagtgcaaagtgtccaacaaggcgctgccggcaccgatcgagaaaactatctccaaagcgaagggacagcctagggaacctcaagtctacacgctgccaccatcacgggatgaactgactaagaatcaagtctcactgacttgtctggtgaaggggttttaccctagcgacattgccgtggagtgggaatccaacggccagccagagaacaactacaagactacccctccagtgctcgactcggatggatcgttcttcctttactcgaagctcaccgtggataagtcccggtggcagcagggaaacgtgttctcctgctcggtgatgcatgaagccctccataaccactatacccaaaagtcgctgtccctgtcgccgggaaag (Human IgG1 light chain DNA variant 1)SEQ ID NO: 25gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc(Human IgG4P heavy chain DNA variant 1) SEQ ID NO: 26caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagatctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacctgggaagggccttgagtggatgggtctggtcgatcccgaggacggcgaaactatctacgccgagaagttccagggtcgcgtcaccatcaccgccgacacttccaccgacaccgcgtacatggagctgtccagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagctactacccgaaccacttcgactactggggacagggcactctcgtgactgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag (Human IgG4P light chain DNA variant 1) SEQ ID NO: 27gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc(Mouse full length IgG1 heavy chain variant 2) SEQ ID NO: 28QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (Mouse full length IgG1 light chain variant 2)SEQ ID NO: 29DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(Human full length IgG1 heavy chain variant 1) SEQ ID NO: 30QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Human full length IgG1 light chain variant 1) SEQ ID NO: 31DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fab heavy chain variant 2)SEQ ID NO: 32QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (Fab light chain variant 2)SEQ ID NO: 33DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Human IgG4P heavy chain variant 2)SEQ ID NO: 34QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Human IgG4P light chain variant 2)SEQ ID NO: 35DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

REFERENCES

Bostrom, M. P. & Seigerman, D. A. (2005), HSS journal: TheMusculoskeletal Journal of Hospital for Special Surgery 1, 9-18. Theclinical use of allografts, demineralized bone matrices, synthetic bonegraft substitutes and osteoinductive growth factors: a survey study.

Buza, J. A., 3rd & Einhorn, T. (2016), Clinical cases in mineral andbone metabolism: The Official Journal of the Italian Society ofOsteoporosis, Mineral Metabolism, and Skeletal Diseases 13, 101-105.Bone healing in 2016.

Canalis, E., Parker, K. & Zanotti, S. (2012), J. Cell Physiol 227,269-277. Gremlin1 is required for skeletal development and postnatalskeletal homeostasis.

Cho, T. J., Gerstenfeld, L. C. & Einhorn, T. A. (2002), Journal of Boneand Mineral Research: The Official Journal of the American Society forBone and Mineral Research 17, 513-520. Differential temporal expressionof members of the transforming growth factor beta superfamily duringmurine fracture healing.

Einhorn, T. A. & Gerstenfeld, L. C. (2015), Nat. Rev. Rheumatol. 11,45-54. Fracture healing: mechanisms and interventions.

Ferguson, C., Alpern, E., Miclau, T. & Helms, J. A. (1999), Mechanismsof development 87, 57-66. Does adult fracture repair recapitulateembryonic skeletal formation?

Gazzerro, E. et al. (2005), Endocrinology 146, 655-665. Skeletaloverexpression of gremlin impairs bone formation and causes osteopenia.

Gazzerro, E. et al. (2007), J. Biol.Chem. 282, 31549-31557. Conditionaldeletion of gremlin causes a transient increase in bone formation andbone mass.

Goulet, J. A., Senunas, L. E., DeSilva, G. L. & Greenfield, M. L.(1997), Clinical Orthopaedics and Related Research, 76-81. Autogenousiliac crest bone graft. Complications and functional assessment.

Hsu, D. R., Economides, A. N., Wang, X., Eimon, P. M. & Harland, R. M.(1998), Mol. Cell 1, 673-683. The Xenopus dorsalizing factor Gremlinidentifies a novel family of secreted proteins that antagonize BMPactivities.

Sato, K., Watanabe, Y., Harada, N., Abe, S., Matsushita, T., Yamanaka,K., Kaneko, T., & Sakai, Y. (2014) Tissue Eng Part C. Methods 20,1037-1041.

Schmid, G. J., Kobayashi, C., Sandell, L. J. & Ornitz, D. M. (2009),Developmental Dynamics: An Official Publication of the AmericanAssociation of Anatomists 238, 766-774. Fibroblast growth factorexpression during skeletal fracture healing in mice.

Yu, Y. Y. et al. (2010), Bone 46, 841-851. Immunolocalization of BMPs,BMP antagonists, receptors, and effectors during fracture repair.

The invention claimed is:
 1. A method for the treatment of a bonefracture or bone defect comprising administering a therapeuticallyeffective amount of an anti-gremlin-1 antibody or functionally activefragment, variant or derivative thereof comprising a heavy chainvariable region comprising SEQ ID NO: 3 or 4 for heavy chain CDR (HCDR)1, SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3 and a light chainvariable region comprising SEQ ID NO: 7 for light chain CDR (LCDR) 1,SEQ ID NO: 8 for LCDR2 and SEQ ID NO: 9 for LCDR3, wherein the bonefracture or bone defect is a delayed-union fracture, a non-unionfracture, a bone defect with a loss of bone, or a bone disease selectedfrom the group consisting of osteoporosis, osteogenesis imperfecta, andPaget's disease of bone.
 2. The method according to claim 1, wherein theanti-gremlin-1 antibody or functionally active fragment, variant orderivative thereof comprises a heavy chain variable region comprisingSEQ ID NO: 3 for heavy chain CDR (HCDR) 1, SEQ ID NO: 5 for HCDR2, SEQID NO: 6 for HCDR3 and a light chain variable region comprising SEQ IDNO: 7 for light chain CDR (LCDR) 1, SEQ ID NO: 8 for LCDR2 and SEQ IDNO: 9 for LCDR3.
 3. The method according to claim 1, wherein theanti-gremlin-1 antibody or functionally active fragment, variant orderivative thereof comprises a heavy chain variable region comprisingSEQ ID NO: 4 for heavy chain CDR (HCDR) 1, SEQ ID NO: 5 for HCDR2, SEQID NO: 6 for HCDR3 and a light chain variable region comprising SEQ IDNO: 7 for light chain CDR (LCDR) 1, SEQ ID NO: 8 for LCDR2 and SEQ IDNO: 9 for LCDR3.
 4. The method according to claim 1, wherein theantibody comprises a heavy chain variable region (HCVR) of SEQ ID NO: 10and/or a light chain variable region (LCVR) of SEQ ID NO:
 11. 5. Themethod according to claim 1, wherein the heavy chain variable regioncomprises a sequence having at least 95% identity to the sequence of SEQID NO: 10 and the light chain variable region comprises a sequencehaving at least 95% identity to the sequence of SEQ ID NO:
 11. 6. Themethod according to claim 1, wherein the functionally active fragment isa Fab, Fab', F(ab')2, Fv or scFv.
 7. The method according to claim 1,wherein the bone fracture or bone defect is a delayed-union fracture. 8.The method according to claim 1, wherein the bone fracture or bonedefect is a non-union fracture.
 9. The method according to claim 1,wherein the bone fracture or bone defect is a bone defect with a loss ofbone.
 10. The method according to claim 1, wherein the bone fracture orbone defect is osteoporosis.
 11. The method according to claim 1,wherein the bone fracture or bone defect is osteogenesis imperfecta. 12.The method according to claim 1, wherein the bone fracture or bonedefect is Paget's disease of bone.